It is thought that within the marrow there are at least two anatomically and physiologically distinct hematopoietic niches; the osteoblastic and the vascular. Within these niches, numerous molecules and secreted factors have been implicated in the strictly regulated process of hematopoiesis. Nevertheless, a thorough understanding of the subpopulations of HSC that interact with each niche and the physiological role of each niche type in the regulation of stem cell hierarchy and function is still missing. Here, we will identify the cellular interactions that occur within each of the niches at the phenotypic, functional, and transcriptional levels, using as a distinct paradigm the developmental process whereby the marrow acquires the ability to support hematopoiesis. Experiments to be performed will employ a physiologically relevant large animal model, high resolution imaging/capture technology, sensitive transcriptome sequencing, and functional studies, which combined provide us with the unique ability to study and manipulate the interactions of human HSC and specific niche cells in vivo, thereby unraveling the role played by each discrete microenvironment in the establishment and maintenance of human hematopoiesis. The overall hypothesis of these studies is that the developmental process whereby the fetal marrow acquires the ability to support hematopoiesis can be used as a model for understanding both the interactions that occur between HSC and the cells comprising the marrow niches, and the role of these niche elements in the initiation/maintenance of hematopoiesis. We further hypothesize that this knowledge will provide the necessary tools for manipulating these niches to facilitate the engraftment and accelerate hematopoietic recover of donor HSC after HSCTx. We propose the following Specific Aims:1)Define the steps and key cellular players in the emergence of the fetal BM hematopoietic niche and in the onset of marrow hematopoiesis, as a tool to understand the cellular interactions that occur both within and amongst the vascular and the osteoblastic niches as they arise, and characterize the nature of the hematopoietic cells each supports, thus delineating the role each plays in the maintenance and expansion of the stem cell pool to drive lifelong hematopoiesis;2) Determine whether the vascular/perivascular niches present within the nascent marrow not only serve as a supportive site for lodging of HSC arriving from the fetal liver, but also harbor cells with the potential to contribute to the establishment of hematopoiesis; and 3) Investigate whether the composition of the bone and vascular niches can be manipulated by specific populations of donor-derived cells to increase the levels of engraftment and/or accelerate reconstitution of donor derived HSC following allogeneic transplantation. Success of the proposed studies would have significant implications in the isolation and expansion of primitive HSC and the understanding of the origin of hematologic malignancies, and could significantly improve the safety and success of clinical HSC transplantation and potentially allow its application to a wider range of diseases affecting human patients.